Discovery Genomics, Inc. is focused on development of the Sleeping Beauty (SB) transposon system as a non-viral means of integrating new gene sequences in cells and tissues for therapeutic purposes. Here we propose to develop a method for improved targeting of the SB system to specific cell types following systemic injection by coupling a targeting tether to the plasmid carrier of the transposon. This approach will allow 'universal' vectors that could carry any gene and be directed to any cell type without modification of the transposase or transposon for each cellular target. This project will dovetail with DGI's ongoing SBIR project to deliver Factor VIII and IX genes in appropriate animal model systems. Thus, this project has high-impact potential because the techniques we will develop can be used for treatment of multiple diseases using a 'universal' non-viral vector system whose properties will be applicable to many therapeutic transgenes. The Specific Aims of the project are to: 1. Construct a Liver- directed Targeting Tether (LTT) vector system to increase uptake of SB transposons into liver cells. This aim will be accomplished by construction of a LTT targeting tether comprised of a LexA DNA-binding domain fused to a ligand specific for hepatocytes and construction of a plasmid, pKLAT2, that contains an SB transposon plus multimeric LexA operator sites to which the targeting tether can bind. 2: Evaluate the efficiency in cultured liver cells (HuH7 and HepG2) of a LTT targeting tether to enhance uptake and transposition of a pKLAT2 transposon vector. HeLa cells will serve as a control for cells that lack appropriate liver-specific receptors. 3: Evaluate the efficiency of a LTT targeting tether to enhance uptake and transposition of a pKLAT2 transposon vector into liver cells in mice. [unreadable] [unreadable] [unreadable]